The invention relates generally to optical chemical and biochemical sensors and more particularly to solid-state sensors using single-step competitive immunoassays with antibodies. For the purposes of this invention the term sensor is used to include all solid state systems capable of doing a single-step competitive immunological analysis where the optical output provides qualitative and quantitative information about the target molecule(s), i.e., sample.
In a single-step competitive immunoassay optical sensor, an antibody is immobilized on the sensor substrate, and a tagged antigen is bound to the antibody. The tag is typically a fluorophore or chromophore. The target (untagged) antigen competitively binds to the antibody and displaces the tagged antigen, causing a change in sensor optical properties, e.g. fluorescence or color intensity. The antibody-antigen reaction is used to identify the measured species and the change in optical properties can be directly related to the concentration of that species. U.S. Pat. No. 4,321,057 issued Mar. 23, 1983 to Buckles describes a fiber optic sensor based on competitive immunoassay between tagged and untagged antigen. It is also possible to adapt the solid-state, single-step immunoassay system so that it can be used with a radio-chemical tag.
Immunoassays have become a well accepted method of analysis in medicine because of their unquestionable specificity to the target compound of interest. If a monoclonal antibody is used, then its reaction is specific to a particular antigen (compound of interest). If, on the other hand, a polyclonal antibody is used, then its reaction is specific to a particular chemical structure rather than a precise chemical compound. A comparison of the types of antibodies suitable for the single-step, solid state competitive immunoassay are listed in FIG. 1A.
The use of immunoassays can be extended beyond basic medical applications to a variety of other analytical needs including environmental monitoring, process control, dosimetry/personal protection and military applications. The solid-state, single step technology described herein is not only applicable to all of these, but extends the number and types of medical applications while simplifying the process.
The preferred existing immunoassay techniques are enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immuno-fluorescent assay (ELIFA), both of which are liquid systems based on the competition between sample analyte and analyte-enzyme conjugates for a limited number of antibody sites. These are multiple step chemical reactions which must be executed with precise timing for meaningful semi-quantitative measurements.
FIG. 1B shows the typical ELISA procedures used in field kits. They involve numerous solution changes, washings, timed reactions, and a series of critical steps that can be a source of operator error, especially under non-optimal field conditions. In this order of events, the following takes place:
1) A filtered liquid sample or a filtered extracted solid sample is added to a tube containing an immobilized antibody specific to the analyte of interest. PA1 2) This is allowed to react for a specific time, the contents of the tube are removed, and the tube is washed. PA1 3) An enzyme-bound solution of pure analyte is added to the tube and allowed to compete for a fixed time, the contents of the tube are removed and the tube is washed. There is now a mixture of analyte (sample) and enzyme-bound analyte attached to the tube in proportion to their concentrations. PA1 4) A chromophore, which reacts with the enzyme, is added to the tube and the color is allowed to develop. PA1 5) After a specific time, a retardant is added to stop color formation. PA1 6) The color intensity is then measured in a field spectrometer (the measurement is made across the tube and background contributions are included in the results).
In actuality three (3) similar reactions are run simultaneously--low analyte, high analyte and blank (reference) and their colors compared to get a semi-quantitative measurement of sample concentration.
The immunosensor technique described herein is also based on a competitive assay. It uses a new, unique single step biochemical approach which not only is much simpler than ELISA or ELIFA, but can be done in the solid state, i.e., no wet chemistry-biochemistry.
This invention primarily addresses comprehensive new approaches to competitive binding fluoro- and chromoimmunoassays. In particular it focuses on simplifying the analyses, optimizing the physical state of the sensor, improving sensitivity, making the sensor quantitative and adapting the sensor for the analysis of small molecules.
The competitive immunoassay of this invention reduces the number of individual analytical steps from an average of six (6) to ten (10) using ELISA or ELIFA to one (1) using this invention. A mechanism has been devised whereby all of the chemistry-biochemistry necessary to perform the detection, identification and quantification functions are inherently a part of the analytical strategy and all of the requisite data are easily accessed. A comparison of typical competitive immunoassay techniques appears in FIG. 1C, where the last column, FCIE, is the present invention. The chemistry-biochemistry of the present invention is innovative with respect to prior art in competitive immunoassays in that it is solid state, i.e., the need for, and use, of a liquid system has been obviated. The chemistry-biochemistry is immobilized directly on a substrate. This substrate can be of any configuration which permits an optical measurement to be made. Substrates include, but are not limited to, test tubes, cuvettes, fiber optics, optical waveguides, optical chips and optical waveguides on semiconductor chips.
Sensitivity is enhanced in the present invention by reducing background contributions. The techniques of background reduction can only be used in the solid-state configuration where all of the components are affixed in a predetermined position and the number of uncontrolled parameters are minimized or completely eliminated. Background reduction is primarily accomplished by designing the chemical-biochemical system so that the fluorescent or chromophoric tag which is ejected during the competitive step in the analysis cannot be and is not in the field-of view of the optical (spectral) measurement. Other methods of minimizing background include blocking the surface of the substrate so that nothing but the antibody can attach to it, assuring that the tag is covalently bonded to the chemistry-biochemistry so that its motion is restricted and using an optical isolation compound to diminish reflections from the sensor's surface.
The use of competitive antibody-antigen reactions has been primarily limited because of: (1) The use of liquid chemical-biochemical systems which inherently include all of the drawbacks and potential human errors associated with wet chemistry-biochemistry; (2) The use of multiple-step analysis--six (6) to ten (10) precise chemical-biochemical steps in a predescribed timed sequence; (3) Lack of sensitivity--high background noise, inadequate antibody loading on the substrate and inefficacious exchange between antibody bound to tagged antigen and untagged antigen and (4) The inability to measure small molecules--a requisite for many environmental, process control, and dosimeter applications.
For the molecules which can be measured using existing techniques such as ELISA, ELIFA or mass spectrometry, the minimum detection limits (MDL) are nominally between 35 and 50 parts-per-billion (ppb), and in some cases as high as the low parts-per-million (ppm) range. The limit of quantification (LOQ) is a factor of 3.3 higher. These restrictions limit or exclude their use in such important areas as: (1) Environmental monitoring, especially measuring pollutants to assure that drinking water, occupational health and safety, and Underwriter Laboratory (UL) standards are met; (2) Measuring contamination in chemical processes; (3) Examining personnel for alcohol, drug or other substances abuse; (4) Determining exposure to and presence of toxic substances and infectious diseases; and (5) Diagnosing and evaluating maladies such as cancer, heart infarctions, arthritis, gastrointestinal ailments, abnormal blood panels and urological problems. Typically, 5 ppb is the LOQ required for many of these applications which means that a MDL of 1.5 ppb is a requisite. The solid-state, single-step competitive immunoassay described herein can have a MDL of less than 0.4 ppb (&lt;400 pptr, parts-per-trillion).
The production of solid-state, single-step, high sensitivity competitive immunoassays for an extended list of antigens is the primary focus of this invention. This provides the ability to measure and quantify numerous antigens in the low ppb to parts per trillion (pptr) range irrespective of their molecular size. Thus the analysis of small molecules is also part of this invention.
As a result of the solid-state, single-step, high-sensitivity chemical-biochemical systems developed according to the present invention, the drawbacks that existing, prior art, immunoassays must be done by trained personnel and are subject to human error, is overcome. Specifically, the up-to-date prior art assays require mixing of chemicals, such as the addition of enzymes and dyes, in exact quantities and sequence, and at designated time intervals. The results, therefore, are only as accurate as the technician and are subject to the sum of all errors. The use of immunological systems, where there is no human participation in the chemistry-biochemistry, is an important part of this invention.
Optical waveguide chemical sensors (OWCS), optical waveguide biochemical sensors (OWBS), fiber optic chemical sensors (FOCS), fiber optic biochemical sensors (FOBS), optical chip chemical sensors (OCCS) and optical chip biochemical sensors (OCBS) as well as simple containers such as cuvettes, test tubes and bottles which can transmit an optical signal are all transducers in an information acquisition strategy which obtains real-time data about the presence and concentration of specific species, or chemical groups of compounds, in chemical and biochemical systems. Optical waveguides include flat channeled and non-channeled waveguides as well as chips with waveguides on them. Waveguide sensors can have a wide variety of general configurations, for example, similar in physical layout to those illustrated in FIGS. 18A-D. A typical waveguide system has a sensor chemistry attached to a portion of the waveguide. More than one sensing chemistry can be placed on a single waveguide. The sensor can be a miniaturized waveguide which is totally covered with sensing chemistry. In order to have an internal reference, the waveguide may be half coated and the uncoated section is used to obtain a reference signal.
Preferably, the waveguide is half coated with the complete immobilization agent, sensing biochemistry and optional overcoatings, and the other half of the waveguide is coated with all but the sensing chemistry and is used to obtain a reference. In this configuration, in order for this to work, the inactive portion of the waveguide must face the illumination source and the reference signal must be taken before that of the coated section. A more practical arrangement is to use two (2) waveguides, sense and reference, illuminated by a single light source and the resultant signal detected by two (2) matched detectors. It is possible to use waveguide sensors with many configurations in combination with a separate reference. In either arrangement the difference or ratio of the sense and reference signal contains the unadulterated concentration information. In the case of container-based sensors the sensing chemistry is attached to the inner wall and the sample to be measured placed in the vessel. As with the waveguide sensors, sense and reference sensors can be employed either by using two vessels, sense and reference, or by attaching both the sense and reference chemistries in the same container in a geometric arrangement that allows each chemistry to be interrogated individually.
Optical chemical and biochemical sensors are devices with indicators for preselected chemical and/or physical properties attached to their surfaces, so that sensitive, specific, real-time analyses can be made. These can be based on fluorescence, absorption, Raman, polarization, refraction, reflection or radiochemical measurements. The species or group-specific chemistry can be selected from organics, inorganics, metals, enzymes, monoclonal and polyclonal antibodies, biochemicals and polymers or combinations thereof. Interaction of an analyte with the sensing reagent (in this case a tagged antigen or tagged antibody) produces a change in one of the above mentioned spectroscopic parameters. For sensitive measurements using antibody-antigen reactions fluorescence, color, or polarization are the preferred measured properties depending on the molecular size of the target molecule. A readout device electronically converts light flux into voltage. Modulation in the voltage reading directly correlates with the analyte concentration.
The basic reactions in a competitive immunoassay are generally similar to the reactions shown in FIGS. 2A,B. In this Figure Y represents an antibody, *.gradient. (or .gradient.*) is the tagged antigen and .gradient. is the untagged antigen, i.e., the target compound of interest. The greater the exchange rate between .gradient. and *.gradient. the more sensitive the reaction. Ideally, most of the *.gradient. will be lost at the actual (or integrated) concentration of the compound to be measured. This is not the case under normal circumstances; however, the present invention provides a method of making this happen.
An antibody (Y) can be attached to a glass or waveguide substrate and saturated with a tagged antigen (*.gradient.), similar to that shown in FIG. 3A. The same arrangement can be produced on a membrane substrate, similar to that shown in FIG. 4A.